It is desirable to make efficient and qualitatively accurate determinations of microscopic amounts of constituents of samples, such as the determination of analytes including hormones, enzymes, metabolites, and drugs in a sample of blood or other fluid. Immunoassay techniques have been used for such determinations.
Immunoassay (IA) is the generic term for systems of quantitative in vitro measurement based on the principal of saturation analysis, displacement analysis, or competitive analyte binding. Since such techniques are extremely sensitive and very specific. IA techniques are used for the determination of physiologically, pharmocologically or forensically important analytes in biological fluids.
One of the techniques in IA is derived from the observation that unlabeled analyte displaces labeled analyte from an antibody that can bind the analyte. With an antibody concentration and the labeled analyte held constant, the binding of the label is quantitatively related to the amount of unlabeled analyte that is added. Thus, known analyte standards can be used initially to prepare a plot of the fraction of bound radioactive analyte against the concentration of added non-labeled analyte.
In operation, the method of heterogeneous immunoassays requires the separation of a labeled analyte of interest into bound and unbound fractions after its interaction with an antibody in the presence of an unknown quantity of unlabeled analyte. Homogeneous assays that do not require a separation step are becoming increasingly popular.
Various devices are used in IA techniques. Occasionally, a microtiter plate is used, the microtiter plate being a plastic plate including a plurality of rows of wells for containing the sample solutions. For example, an antibody can be bound to the walls of the wells. In radioimmunoassays (RIA), a derivative of the analyte which is radioactively labeled is used as signal generator for quantitative measurements of analyte. A sample or standard and radiolabeled analyte (tracer) are added to the wells The constituents of the wells are agitated during incubation and the liquid of the wells is removed. The wells are then separated and placed in a radioactivity counter to determine radioactive binding. Alternatively, the analyte can be labeled with non-radioactive substances including enzymes and substances emitting fluorescence or luminescence. In addition, the label can be on a second binding substance that binds to the analyte that is already bound to the wall of the well.
Several problems have been uncovered utilizing the aforementioned technique. Standards of the substance to be measured are prepared prior to assaying samples of unknown substance concentration from a stock solution of substance by serial dilutions. These dilutions are then distributed to the designated wells or other containers which are then subjected to the assay procedure. From an obtained physical measurement (e.g. radioactivity), a standard curve is eventually constructed which represents the mathematical or graphical function (standard curve of the concentration of the substance in the standards, that being the known parameter) against the physical measurements of the unknown samples. The standard curve is then used to extrapolate from a physical measurement in a sample of unknown substance concentrations to the concentration predicted by the standard curve.
The disadvantage of this prior art technique involves the preparation of serial dilutions for the standards prior to assaying. This is inefficient because it is time consuming and can introduce inaccuracies by the operator. Each serial dilution allows for a first inaccuracy to be developed and each following serial dilution perpetuates and sometimes accentuates the inaccuracy. Therefore, there are prior art techniques utilizing predispensed standards. However, these standards are distributed such that each standard solution needs to be delivered to the assay well separately with a single pipettor.
A second concern is an efficient means for mixing the contents of each of the wells during the incubation step of the procedure. Care must be taken to not spill the contents of each well yet sufficient agitation must be made in order to properly mix the contents of each well.
Another concern during the processing of the samples of the microtiter plates is that some manufacturers manufacture trays comprising unitary rows of wells interconnected together, each row being connected at its end to a surrounding base of the plate. Once several rows are removed from the plate, it is possible to confuse the order of the rows and thereby lose the identity of tested samples.
The present invention provides an efficient means for providing for transfer a plurality of known concentration standards so that the standards can be transferred in a single step. The present invention further provides an effective and efficient means for agitating the wells containing the standards and samples. The present invention further provides a means of identifying the proper location of a row of wells which has been displaced from the microtiter plate to avoid loss of identity of the row. Finally, the present invention provides a single kit within a single housing incorporating the aforementioned inventions into a unitary kit providing all of the means necessary to conduct an IA incubation procedure.